This Small Business Innovation Research Phase I proposal develops a technology that allows bioactive proteins to be synthesized and secreted from cultured plant cells in high yields and with prolonged serum half-life. The innovation of this technology, designated Hyp-Glyco, involves the expression of secreted therapeutic proteins as fusions with a novel hydroxyproline (Hyp)-Glyco tag. The Hyp-Glyco tag also extends serum half-life and facilitates purification of secreted proteins while retaining protein bioactivity. This proposal is focused on scale-up process development of Hyp-Glyco technology in bioreactor cultured tobacco cells expressing interferon 2b and in development of a cost-effective technique for protein recovery/purification. Specific aims of this feasibility Phase I are to 1) Establish bioreactor based high yield interferon protein production in plant cell cultures using Hyp-Glyco technology;2) Demonstrate that continuous perfusion culture technology will further enhance protein production;and 3) Show that reliable and cost-effective recovery and purification of bioactive interferon protein can be achieved in bioreactor grown plant cell cultures. Success of Phase I will support a follow-up Phase II aimed at establishing cost-effective commercial level production protocols for all phases of this new protein production technology so as to provide the confidence necessary for investing in Phase III commercial development. Protein therapeutics represents a multi-billion-dollar marketplace, but costly manufacturing methods for these drugs currently make them prohibitive for consumers. The broad impact of the proposed work resides in the potential of an alternative less costly platform for enhanced production of therapeutic proteins in plant cells which would, simultaneously, provide the added advantage of improved clinic effectiveness. Because this technology not only produces high-yield secreted proteins but also eliminates the time-consuming and expensive process of chemical derivatization to improve clinic effectiveness, the production costs can be significantly reduced. With the additional safety advantage of substituting plant cells for existing animal cell production, "molecular farming" could become commercially competitive with current mammalian and microbial cell production systems. Importantly, the interferon with extended serum half-life described here would meet a significant demand from 4 million hepatitis C virus (HCV) infected patients in USA and 3% of population worldwide. This technology is also broadly applicable to a wider range of therapeutically important proteins such as hGH, G-CSF and EPO etc, all of which represent billion-dollar markets. The development and success in commercializing the Hyp-Glyco protein manufacturing technology would create job opportunities in economically disadvantaged regions of the US and drive local economic development. PUBLIC HEALTH RELEVANCE: The growing dual threat of environmental pollution and global warming is considered by most health/medical experts and governments as one of the most dangerous and intransigent potential health problems globally. The need for developing new health positive, cost conscious and environmentally sustainable technologies for production of industrial and pharmaceutical products is a high priority of health related research in the U.S. and abroad. This proposal will target this need directly by developing and testing the feasibility of innovative more cost-effective methods for producing therapeutic proteins by using plant-based cultures and bioreactors which will confer the simultaneously benefits of greater safety, energy conservative and environmental sustainability than traditional manufacturing methods.